Fundamental to pharmaceutical research and development is an early determination of whether a particular proposed agent presents a hazard of mutagenicity, or genotoxicity. It is generally accepted that the mutagenic potential of a particular agent, e.g., compound, is roughly proportional to its carcinogenic potential.
Mutagens are agents, such as chemical carcinogens, that cause an increase in the rate of mutation. A mutation is generally thought of as a change in the DNA sequence of an organism due to, e.g., gene or point mutations, primary DNA damage and repair, or chromosomal alterations.
Commonly employed tests for detecting mutagens or genotoxins (mutagenicity tests) include, for example, the Salmonella mutagenicity test (Ames Test, Ames et al., 1973a, 1973b, and 1975; see also, Ames, B. N., 1971), and the more recent Salmonella or Escherichia coli lactam mutagenicity tests (β-lac tests, Lee, C -C, et al., 1994, and Hour, T -C., et al., 1995). Both of these tests are based on the ability of DNA damaging agents to produce reverse mutations in certain bacterial genes. The Salmonella mutagenicity test uses Salmonella strains that each contain a different type of mutation in the his (histidine biosynthesis) operon, e.g., frameshift or base pair substitutions. The Salmonella or Escherichia coli (E. col) strains used in the lactam mutagenicity test contain a plasmid which itself contains a β-lactamase gene site having a mutation precluding β-lactamase expression. The β-lactamase gene encodes a protein that hydrolyses amide bonds in β-lactam rings of penicillins and Cephalosporins to derivatives devoid of antimicrobial activity, thus rendering such microbes able to resist antibiotics of the β-lactam class.
This Salmonella test system, also known as the Salmonella/mammalian microsome mutagenicity assay, allows for the screening of suspected carcinogens, the isolation of carcinogens from natural materials, and the identification of the active forms of carcinogens. The Ames Test, the most widely used and investigated mutagenicity assay (see, for example, Maron, D. M. and Ames, B. N., 1983; Dunkel, V. C. et al., 1985; and Zeiger, E., 1985; and Kier, L. E. et al., 1986), remains the recommended assay for bacterial mutagenesis.
Variations of the Salmonella/mammalian microsome mutagenicity assay continue to be reported by Ames and others (see, for example, Yahagi, T. et al., 1975; Prival, M. J. and Mitchell, V. D. 1982; Haworth, S. et al., 1983; Kado, N. Y., et al., 1983; and Reid, T. M., et al., 1984; and Current Protocols in Toxicology, John Wiley & Sons, Inc. (2000), Chapter 3. Genetic Toxicology: Mutagenesis and Adduct Formation, Chapter 3 Introduction, Unit 3.1 The Salmonella (Ames) Test for Mutagenicity, Alternate Protocol 1: Plate Assay With Preincubation Procedure; Alternate Protocol 2: Desiccator Assay for Volatile Liquids; Alternate Protocol 3: Desiccator Assay for Gases; Alternate Protocol 4: Reductive Metabolism Assay; Alternate Protocol 5: Modified (Kado) Microsuspension Assay).
Several of these modifications have generally focused on minimizing the required amount of test agent and increasing throughput, i.e., minimizing the manual work of the Ames Test (standard plate-incorporation whereby the bacterial tester strain is exposed to incremental doses of the test agent in the presence of an exogenous metabolic activation system) (see, for example, Waleh, N. S. et al., 1982; and Current Protocols in Toxicology, Chapter 3. Genetic Toxicology: Mutagenesis and Adduct Formation, Chapter 3 Introduction, Unit 3.1 The Salmonella (Ames) Test for Mutagenicity, Support Protocol 1: Toxicity Test for Dose Selection).
A revised protocol (preincubation assay) that deviates from Ames' standard agar plate incorporation has been described (Maron, D. M. and Ames, B. N., 1983). Another variation, Mutascreen®, combines turbidimetric and kinetic principles into a bacterial mutagenicity test based on the same biological system as in the Ames Test (Falck, K., et al., 1985).
The spiral Salmonella assay, another automated approach to bacterial mutagenicity testing, reportedly eliminates the need for serial dilutions and multiple plates to obtain the dose-response data (see, for example, DeFlora, S., 1981; Couse, N. L. & King, J. W., 1982; Houk, V. S. et al., 1989, and 1991).
Other methods retain the use of solid agar plating, such as, for example, the automated liquid preincubation exposure protocol described by Kato, H. et al., 1995.
The Miniscreen, a scaled-down version of the Ames Test, describes the use of smaller quantities of test agent (20 mg) versus the 2 g of test agent reportedly required by the Ames test, and is used as a blanket or pre-screen for various types of test agents (Brooks, T. M., 1995; see also, Burke, D. A. et al., 1996).
Further, the Ames II™ test (Xenometrix) is described as a modification to the fluctuation assay (Green, M. H. L. et al., 1976; Gatehouse, D. G. & Delow, G. F., 1979; and McPherson, M. F. & Nestmann, E. R., 1990) to allow automation of plating the exposed cells in selective media using the TA7000 series of tester strains.
Despite the relatively continued provision to the art of such modified protocols, for example, as provided above, the Ames Test remains the recommended assay for bacterial mutagenesis; hence, those skilled in the art will appreciate that there remains a need for assays that further overcome the limitations of the original and modified versions of the assay, such as, for example, the amount of test agent required, the amount of time it takes to reach a result, manual counting of revertant colonies, the lack of ease of scalability to high throughput assays, and the like.
Likewise, those skilled in the art will understand that similar limitations exist in relation to the known β-lactam tests, which measure reverse mutation from ampicillin-sensitivity to ampicillin-resistance (see, for example, Bosworth, D. et al., 1987; Foster, P. L. et al., 1987; Delaire, M. et al., 1991; Lee, C -C., et al., 1994; and Hour, T -C., et al., 1998), as well as the known mutagenicity assay premised on the point mutation in a tryptophan gene (e.g., using E. coli WP2, see, for example, McCalla, D. R. and Voutsinos, D., 1974).
The present invention provides, in part, improved reverse mutagenicity assays. The assays preferably employ the well characterized Salmonella typhimurium strains utilized in the Ames Test, except that these strains have been further modified as described herein to contain a plasmid comprising an expressible heterologous lux(CDABE) gene complex under the control of a constitutive promoter. This is to empower these microorganisms to emit light as a readout when metabolically active as described in more detail hereinbelow.
Bacterial bioluminescence and lux operons, as well as applications thereof, are well known in the art (see, for example, Meighen, E. A., 1988; Frackman, S. et al., 1990; Jassim, S. A. A. et al., 1990; Stewart, G. S. A. B., 1990; Stewart, G. S. A. B. & Williams, P., 1992; Meighen, E. A., 1993; Hill, P. J., et al., 1993; Bronstein, I. et al., 1994; Hill, P. J. & Stewart, G. S. A. B, 1994; Marincs, F. & White, D. W. R., 1994; Chatterjee, J. & Meighen, E. A., 1995; and Voisey, C. R. & Marincs, F., 1998).
The luxA and luxB genes of the lux structural operon encode the non-identical α and β subunits of a bacterial luciferase, respectively, and are widely used as reporter genes (see, for example, Stewart, G. S. A. B. & Williams, P., 1992; and Chatterjee, J. & Meighen, E. A., 1995). The resultant heterodimer catalyzes the oxidation of FMNH2 and a long-chain fatty aldehyde, which results in an emission of light. Metabolically active bacterial cells produce FMNH2,
The luxC, luxD, and luxE genes of the lux operon encode the fatty reductase complex, where luxC encodes the reductase polypeptide, luxD encodes the transferase polypeptide, and luxE encodes the synthetase polypeptide. The fatty acid reductase complex produces the aldehyde substrate necessary for the luciferase. The cleavage of the substrate by the luciferase requires endogenous FMNH2 and produces bioluminescence.
The bacterial bioluminescence reaction also requires endogenous FMNH2 and O2. As described above, metabolically active bacterial cells produce FMNH2. Metabolically active cells of the novel bacterial tester strains will be those that, after exposure to a test agent, revert to a non-mutant phenotype, and thus are able to grow in a selective medium, e.g., a medium not containing histidine, a medium not comprising tryptophan, a medium comprising ampicillin, as the case may be, and due to such growth (or metabolic activity) emit luminescence, in an amount greater than the amount, if any, produced by the degree of spontaneous reversion of the mutation, where the degree of spontaneous reversion can be measured, for example, by having additional samples in a given assay that contain, e.g., vehicle (e.g., solvent) and cell only, and/or vehicle and cell and exogenous metabolic activation system. Further, where so desired, the presence of the test agent can be in the presence and absence of the exogenous metabolic activation system.
Depending upon the novel bacterial strain selected, as little as ng/μg amounts of a test agent may be used. In addition, the bioluminescence readout of light-producing revertant colonies is substantially immediate, and due to the constitutive promoter driving the lux(CDABE) expression, maintained as long as the revertant colonies maintain their metabolic activities, e.g., produce FMNH2. Thus, the present invention provides assays that utilize bioluminescence as a sensor for a cell's ability to produce energy. Hence, the present assays can be adapted, based on the present description, as so desired, to any assay using metabolic activity as a readout.
Moreover, the cell for use in the assays of the present invention can be a mammalian cell, which contain a variety of luciferases (see, for example, Bronstein, I., et al., 1994). The most common is luc gene from American firefly (Photinus pyralis). In this system, luciferin serves as substrate for cleavage by the luciferase encoded by the luc gene. Hence, an exogenous substrate, e.g., a luciferin, is supplied, in any suitable manner, to the assays of this invention that utilize mammalian cells. Further, the bioluminescent reaction requires ATP, as source of energy, and oxygen which, as those skilled in the art will appreciate, is abundant in the environment.
The improvements to the Ames Test, and to the modifications thereof, provided by the present invention, further decrease the amount of test agent required and the laboriousness of such protocols, and enable a substantially immediate readout, and thus a determination of whether a particular test agent causes a reversion of a point mutation, i.e., is a mutagen. Such assays readily allow for automation and scale-up for the long sought after assay capability for high throughput screening. Such scalability provides substantial utility to the pharmaceutical industry by enabling high throughput screening of putative pharmaceutical products (for mutagenicity or genotoxicity) at the very early stages of pre-clinical research. The early read on genotoxicity can decrease the aftrition of pharmaceutical product candidates, thus enhancing the efficiency of decision making. Moreover, the assays provided by the present invention can also be used to determine the characteristics, e.g., particular substituents, of compounds that confer genotoxicity to the compound and, as such, assist in the design of non-mutagenic compounds.
All of the documents cited herein, including the foregoing, are incorporated by reference herein in their entireties.